1. Field of the Invention
This invention relates to methods for detecting the presence of target nucleotide sequences.
Nucleic acid hybridization has been employed for investigating the identity of nucleic acids. Hybridization is based on complementary base pairing. When at least partially complementary single stranded nucleic acids are incubated in solution under suitable conditions of temperature, pH and ionic strength, complementary base sequences pair to form double stranded stable hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with its complementary nucleic acid sequence has been employed as an analytical tool in recombinant DNA research. The availability of radioactive nucleoside triphosphates of high specific activity and the synthetic methods of incorporating these nucleotides into nucleic acid probes such as the .sup.32 P labelling of DNA with T.sub.4 polynucleotide kinase and other well known methods has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states mediated by alteration in or additions to nucleic acid composition of the host. These alterations in nucleic acid composition will include genetic or environmental change in DNA by insertions, deletions, rearrangements, point mutations, or acquiring foreign DNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentration of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive rapid method of DNA hybridization analysis.
Current methods for detecting DNA probes generally involve immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labelled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a carefully controlled temperature to remove unbound and nonspecifically bound probe without removing specifically bound probe. The support is then dried and the hybridized material is detected by autoradiography or by colorimetric methods. When very low concentrations must be detected, the ratio of specific to non-specifically bound probe can be very low and repeated washing under highly stringent conditions is frequently required. Under these conditions the sensitivity of the assay is often compromised because of substantial loss of specifically bound probe and failure to achieve a high enough specific to non-specific binding ratio to permit easy detection. When very low concentrations must be detected, the current methods are slow and labor intensive and non-isotopic labels that are less readily detected than radiolabels are frequently not suitable.
The use of .sup.32 P nucleotide as a nucleic acid probe label is not desirable for several reasons. First, .sup.32 P has a relatively short half life of 14.2 days. Consequently, the complementary probe DNA must be prepared just prior to the hybridization procedure to achieve maximum sensitivity. Secondly, the high decay energy of .sup.32 P creates handling and disposal problems and undesirable hazards. It would therefore be advantageous in most cases to utilize a label which is less hazardous and prolongs the shelf life of the probe. High specific activity tritium labelled probes present one alternative. While tritium has not generally been found useful in previous hybridization methods which requires extended exposure times for detection by autoradiography, a solution method which detects labelled probe by liquid scintillation counting would be highly sensitive and desirable. Another less energetic isotope than .sup.32 P but with a long half life is carbon-14.
The development of non-radioactive labeling of nucleic acid probes presents another alternative. A sensitive non-radioactive DNA labelling system is described by Langer, et al., Proc. Nat. Acad. Sci., USA, 78, 6633 (1981). The system is based on the incorporation of a biotinylated deoxyuridine triphosphate into the DNA probe by the nick translation procedure. The resultant biotinylated DNA probe is stable and behaves as does a non-biotinylated DNA probe. Detection of the biotinylated DNA has been applied to the detection of specific DNA and RNA sequences in fixed cells or in tissues following in situ hybridizations and also in hybridizations of DNA fragments separated by gel electrophoresis and transferred onto nitrocellulose filters. The detection of the hybridized biotinylated probe is accomplished by either fluorescent antibody or enzyme amplification techniques. These techniques are further described by Gardner, BioTechniques, 1, 38 (1983) and Lewin, Science, 221, 1167 (1983).
Other non-radioactive methods which have been described involve conjugating a fluorescent molecule such as tetramethylrhodamine or fluorescein isothiocyanate, to the 3'-terminus of single stranded RNA. These fluorescent RNA probes have been applied to cytochemical hybridizations. Bauman, et al., J. Histochem. Cytochem, 29, 227-238 (1981).
A method for increasing sensitivity to permit the use of a simple, rapid, (nonisotopic), homogeneous or heterogeneous method for detecting nucleic acid sequences is necessary.
2. Description of the Prior Art
Langer, et al., Proc. Natl. Acad. Sci. USA, (1981) 78, 6633-6637 discloses the enzymatic synthesis of biotin labelled polynucleotides and the use of these materials as novel nucleic acid affinity probes. The detection of viral genomes in cultured cells and paraffin imbedded tissue sections using biotin labelled hybridization probes is discussed by Brigati, et al., Virology, (1983) 126, 32-50. U.S. Pat. No. 4,486,539 discloses the detection of microbial nucleic acids by a one step sandwich hybridization test. Sensitive tests for malignancies based on DNA detection is described in U.S. Pat. No. 4,490,472. U.S. Pat. No. 4,480,040 discloses the sensitive and rapid diagnosis of plant viroid diseases and viruses employing radioactively labelled DNA that is complementary to the viroid or to the nucleic acid of the virus being diagnosed. European Patent Application 83106112.2 (Priority U.S. Patent Application Ser. No. 391,440 filed Jun. 23, 1982) teaches modified labelled nucleotides and polynucleotides and methods of preparing, utilizing, and detecting the same. Methods and compositions for the detection and determination of cellular DNA are disclosed in U.S. Pat. No. 4,423,153.
Detection and isolation of homologous, repeated and amplified nucleic acid sequences is disclosed in U.S. Pat. No. 4,675,283. A single stranded self-hydridizing nucleic acid probe capable of repeatedly hybridizing to itself or other nucleic acids to form an amplified entity is described in U.S. patent application Ser. No. 888,058, filed Jul. 22, 1986. U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose a homogeneous polynucleotide displacement assay with digestion of the displaced RNA single strand polynucleotide from the reagent complex and amplifying nucleic acid sequences with treatment of separate complementary strands of the nucleic acid with two oligonucleotide primers. European Patent Application No. 0200362 describes a process for amplifying, detecting or cloning nucleic acid sequences and useful in disease diagnosis and in preparation of transformation vectors. A method for simple analysis of relative nucleic acid levels in multiple small samples by cytoplasmic dot hybridization is described in U.S. Pat. No. 4,677,054. A hybridization method of detecting nucleic acid sequences with a probe containing a thionucleotide is described in U.S. Pat. No. 4,647,529.
A simple and efficient enzymatic method for covalent attachment of DNA to cellulose and its application for hydridization-restriction analysis and for in vitro synthesis of DNA probes is described in Nucleic Acids Research (1986) 14:9171-9191. Cleavage of single stranded oligonucleotides by Eco RI restriction endonuclease is described in Nucleic Acid Research (1987) 15:709-716.
A nucleic acid hybridization assay employing probes cross-linkable to target sequences is described in U.S. Pat. No. 4,599,303. The method involves the preparation of a specific single stranded ribonucleic acid or deoxyribonucleic acid molecule into which a bifunctional cross-linking molecule has been covalently incorporated. The incorporation is such that the cross-linking molecule retains the capacity to undergo a second reaction with the nucleic acid of the bacterial, viral, or mammalian chromosome, which is the target for the probe such as to form a covalent cross link. Following cross-linking, the uncrossed link probe is separated from covalently cross-linked probe-target complex using one of several procedures which differentiate between single stranded probe and double stranded covalently linked probe-target complex.
Detection of target sequences in nucleic acids by hybridization using diagnostic and contiguous probes for diagnosis of genetic abnormality diseases, especially in an automated procedure, is described in European Patent Application No. 0 185 494A2. In the method a sample is hybridized with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe) and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe) under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe that is complementary to the target sequence and the probes which are not attached are removed. In a preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid target probe duplex, eluting the dissociated target probe, and testing for the label.
The above method suffers at least one disadvantage in that contiguous sequences are required. To carry out the method, one must identify the diagnostic sequence and the contiguous sequence and create diagnostic and contiguous probes complementary to the above sequences. If the diagnostic and contiguous sequences are not identified precisely, then the diagnostic and contiguous probes may not hybridize sufficiently and the assay specificity and sensitivity is lost or substantially decreased.